1. Field of the Invention
The present invention relates to a stimulable phosphor sheet. More particularly, the invention relates to a stimulable phosphor sheet advantageously employable in an autoradiography utilizing a stimulable phosphor sheet.
2. Description of the Prior Arts
There has been heretofore known a radiographic process termed "autoradiography" or "radioautography" comprising steps of: introducing a radioactively labeled substance into an organism; superposing the organism or a part of tissue of the organism (that is, a sample or specimen) on a radiographic film such as a high sensitivity type X-ray film for a certain period of time to expose said film thereto; and obtaining the locational information on the radioactively labeled substance in said sample from the resolved pattern of the film. The autoradiography has been utilized, for example, to investigate the pathway and state of metabolism, absorption, and excretion of the substance introduced in the organism in detail. Such autoradiography is described, for instance, in the following literature: Method in Biochemical Experiment, Volume 6, Method in Tracer Experiment I, 271-289, "8. Autoradiography" by Toru Sueyoshi & Akiyo Shigematsu (Tokyo Kagaku Dozin Ltd., 1977).
The autoradiography has been also utilized to obtain locational information ont he radioactively labeled substances present on a medium containing radioactively labeled tissue of an organism and/or the radioactively labeled substances originating from an organism. For instance, there is known an autoradiography comprising steps of: labeling organism-originating biopolymers such as proteins or nucleic acids with a radioactive element; resolving the mixture of the radioactively labeled biopolymers, derivatives thereof, or cleavage products thereof on a gel support (medium) through a resolving process such as gel electrophoresis; placing the gel support and a high sensitivity X-ray film together in layers for a certain period of time to expose said film to the gel support, developing said film, obtaining the locational information of the radioactively labeled substances from the developed film, and then performing identification of the polymeric substances, determination of molecular weight of the polymeric substances and isolation of the polymeric substances based on the obtained locational information.
As for the autoradiography mentioed above, details are given in A. H. Gordon: ELECTROPHORESIS OF PROTEINS IN POLYACRYLAMIDE AND STARCH GELS, North-Holland Publishing Company, Amsterdam, 1969.
Recently, the autoradiography has been effectively used especially for determining the base sequence of a nucleic acid such as DNA. Therefore, the autoradiography is thought to be a very useful means in the field of structural determination of polymeric substances originating from organisms.
Maxam-Gilbert method and Sanger-Coulson method are known as methods for sequencing DNA utilizing the autoradiography. In these methods, the base sequence of DNA is determined by utilizing a characteristic structure of DNA in that DNA is in the form of a double helix structure consisting of two chain molecules stabilized through hydrogen bonding between two bases of each chain molecule, that the base, which is a part of constitutional unit of DNA, is limited to only four, namely, adenine (A), guanine (G), cytosine (C), and thymine (T), and that the hydrogen bonding between each constitutional base unit comprises only two combinations, namely, G-C and A-T.
For instance, Maxam-Gilbert method is performed by the procedure described below.
A group containing a radioactive isotope of phosphorus (P) is attached to a chain molecule of DNA or a DNA fragment at one end to be sequenced to prepare a radioactively labeled substance, and then the radioactively labeled DNA molecule is specifically cleaved at the specific constitutional unit containing a certain base by a certain chemical reaction This reaction is called a "base specific cleavage reaction". Then a mixture of numerous cleavage products of the DNA or DNA fragment, which is cleaved base-specifically by the above-mentioned procedure is resolved through gel electrophoresis to obtain a resolved pattern, in which numerous cleavage products are resolved depending on the molecular weight, which is approximately proportional to the length of molecule of the cleavage product, to form a band spectrum, or a ladder pattern (the bands are not visible) on the gel medium. The electrophoresed gel is subsequently placed in contact with a high sensitivity X-ray film for a long time at a low temperature, whereby the X-ray film is exposed to the resolved pattern, to cause the radiation from the respective bands containing the radioactively labeled cleavage products to form a latent image of the resolved pattern thereon. The X-ray film having the latent image thereon is developed to obtain a visible band spectrum consisting of a large number of bands which corresponds to the resolved pattern. Then the distance of the each band of the base-specifically cleaved product from the starting position of electrophoresis, which corresponds reversibly to the sequential position from the radioisotopically labeled terminal end of the DNA molecule, is obtained from the developed film. Thereafter, by arranging the bands of the base specific cleavage products of four bases in accordance with the distance obtained by the above-mentioned procedure, the sequential position of each base from the radioisotopically labeled end of the chain molecules is read by referring to the applied base specific chemical reaction.
Maxam-Gilbert method summarized above is described in detail in the following text: METHODS IN ENZYMOLOGY, VOL. 65, PART I (ACADEMIC PRESS, NEW YORK LONDON TORONTO SYDNEY SAN FRANCISCO, 1980)
Sanger-Coulson method also utilizes the specific structure of DNA and is employable for determining the sequence of bases in DNA by the use of DNA synthesis enzyme, gel electrophoresis, and the autoradiographic process.
The characteristics and procedures of Sanger-Coulson method as well as those of the above-mentioned Maxam-Gilbert method are briefly described in the following publicatication: "Reading the genetic information in the original language. A surprising method for sequencing the bases of DNA" written in Japanese by Kin-ichiro Miura, Modern Chemistry, September 1977, pp. 46-54 (Tokyo Kagaku Dozin Ltd., Japan).
As described above, the autoradiograhy is effectively employed for separating or identifying radioactively labeled substances originating from an organism through the steps of: resolving a mixture of the radioactively labeled substances on a support medium (e.g., support medium for electrophoresis, support medium for thin layer chromatography, etc.), and detecting one or two locational information on the resolved substances utilizing their radioactivity. This autoradiographic process is effectively employable, for instance, for determination of structure of biopolymers. For this reason, the present autoradiographic process is also utilized widely.
Nevertheless, such useful autoradiography is not free from several drawbacks in the practical use.
In the first place, a long period of time and complicated operations are involved for performing the procedure of exposing a radiographic film such as a high sensitivity X-ray film to a support medium carrying the radioactively labeled substances resolved thereon to visualize the position of the labeled substances. More in detail, in the conventional autoradiography, the above-mentioned exposing procedure is performed at a low temperature (for instance, in the vicinity of 0.degree. C., or -70.degree. to -90.degree. C. for exposure in the procedure for base-sequencing of a nucleic acid) for a long period of time (for instance, several days). The reasons why these conditions are necessary are that the long exposure time is required to attain an appropriate exposure because the radioactively labeled substances subjected to autoradiography are generally not provided with high radioactivity, and that the photosensitive silver salt of the radiographic film is chemically fogged by various substances contained in the support medium when the film is kept at a relatively high temperature such as room temperature for a long period of time during the exposure, resulting in difficulty of obtaining an exposed image with high accuracy. Thus, the exposure ought to be carried out at a low temperature to suppress chemical fog. It may be proposed that a radiographic film be more sensitized to mitigate the severe exposure condition, but a radiographic film used in the conventional autoradiography is already provided with very high sensitivity, and satisfactory further enhancement in the sensitivity can not be expected, so far as the sharpness of an image to be obtained should be taken into consideration.
In the second place, the photosensitive silver salt of a radiographic film has a drawback that it is sensitive not only to the chemical irritation but also to physical inpetus, and this drawback brings about difficulty in the autoradiographic process and decreases accuracy thereof. More in detail, since the exposing proedure in necessarily carried out keeping a radiographic film in contact with the support medium, the radiographic film is generally handled with no protective cover during operations such as transferring and installing operations for the radiographic film. Accordingly, the radiographic film is likely brought into contact with hands of the operator and tools in the handling, and the physical pressure arising from these contacts causes production of the physical fog on the radiographic film. Thus produced physical fog is also a cause of the decrease of accuracy in the autoradiography. For this reason, the handling of a radiographic film requires well-trained skill and a great caution to avoid to production of the physical fog on the radiographic film, and such careful handling required brings about further complexity into the autoradiographic procedure.
In the third place, certain natural radioactive substances contained in the sample mixture in addition to the radioactively labeled substances participates in the exposure of the radiographic film because the exposure is carried out for a long time in the conventional autoradiography. Thus, the influence of the natural radioactive substances further reduces the accuracy of the locational information of the radioactively labeled substances. In order to remove the troublesome noise brought about by the natural radioactive substances, parallel experiments using control samples and a method for optimization of the exposure time have been employed, but these procedures include increased experimental runs for the parallel experiments and requires preliminary experiments to determine the preferable exposure time, and thus the drawback arising from the compicated procedures not avoidable as a whole.
The present inventors had studied for solving the above-described problems attached to the conventional autoradiography, and discovered that these problems are solved or reduced by using a stimulable phosphor sheet having a phosphor layer comrpising a stimulable phosphor dispersed in a binder as the radiosensitive material in place of the conventional radiographic film, which was applied for patent as U.S. Ser. No. 549,417 and as EP No. 83110984.8.
More in detail, the employment of a stimulable phosphor sheet having a phosphor layer comprising a stimulable phosphor dispersed in a binder as the radiosensitive material for obtaining the locational information of radioactively labeled substances resolved on a support medium in the autoradiography disclosed in the above-mentioned patent applications makes it possible not only to greatly shorten the exposure time required, but also to obtain an accurate locational information on the radioactively labeled substances even under the condition that the exposure is performed at a relatively high temperature such as an ambient temperature or a temperature in the vicinity of the ambient temperature. This fact greatly simplifies the exposing procedure adopted in the conventional autoradiography which should be carried out under chilled condition. Since the exposure time can be greatly shortened, the autoradiographic process can be carried out efficiently in a very short time as a whole. This feature is also very advantageous in the practical operations.
Further, by the employment of the stimulable phosphor sheet in the autoradiography as the radiosensitive material, neither chemical fog nor physical fog, both of which bring about serious problems in the process using the conventional radiographic film, is produced on the obtained image. This also provides advantageous feature in the improvement of accuracy and workability of the autoradiography.
Furthermore, in the case of using the stimulable phosphor sheet as the radiosensitive material, the visualization is not always required to obtain the information of the location of the copied radioactively labeled substance, that is, the locational information can be obtained in desired forms such as a visible image, symbols, numericals and combinations thereof, by scanning the phosphor sheet with an electromagnetic wave such as laser to release at least a portion of radiation energy stored in said phosphor sheet as stimulated emission, and detecting the stimulated emission to obtain locational information on the radioactively labeled substances on the support medium. It is also possible to obtain the required information in desired various forms by further processing the above-mentioned locational information using an appropriate electric means.
As described above, the autoradiography employing the stimulable phosphor sheet has various advantageous features and can be vary effectively utilized.
In the above-mentioned autoradiography, the autoradiographic process can be performed by resolving a mixture of radioactively labeled substances on an independently placed support medium (e.g., support medium for electrophoresis, or support medium for thin layer chromatography) and subsequently superposing the support medium on the stimulable phosphor sheet for performing the exposure. However, in view of the advantageous characteristics of the autoradiography using the stimulable phosphor sheet, it is preferable that the support medium is beforehand provided on the surface of the phosphor sheet and used in this composite form for resolution of the mixture. The detection of the locational information of the resolved substances can be carried out on the stimulable phosphor sheet still carrying the support medium or on the phosphor sheet from which the support medium has been removed.
The support medium can be provided on the surface of the stimulable phosphor sheet by coating an aqueous solution of a polymer or a starting material for the preparation of the polymer, the polymer being chosen from the polymers known as materials of the support medium, over the surface of the stimulable phosphor sheet, and subsequently drying or heating the coated layer. However, since the surface of the stimulable phosphor sheet is generally provided with a protective layer of hydrophobic plastic material which shows poor affinity to the aqueous solution and the material constituting support medium, the satisfactory adhesion between the phosphor sheet and the support medium is hardly accomplished. The poor adhesion between them possibly causes separation of the support medium from the phosphor sheet. Particularly, the poor adhesion is liable to introduce voids between the surfaces of the stimulable phosphor sheet and the support medium, and accordingly the resolution on the support medium is not performed uniformly, to remarkably reduce the accuracy of the autoradiographic analysis. For these reasons, the conventional stimulable phosphor sheet is not sufficiently appropriate for use in the autoradiographic process utilizing the phosphor sheet in the form of a composite consisting essentially of the phosphor sheet and a support medium beforehand superposed thereon.